MUTAGENESIS OF RETROVIRAL VECTORS TRANSDUCING HUMAN BETA-GLOBIN GENE AND BETA-GLOBIN LOCUS-CONTROL REGION DERIVATIVES RESULTS IN STABLE TRANSMISSION OF AN ACTIVE TRANSCRIPTIONAL STRUCTURE

Retrovirus-mediated gene transfer of the human beta-globin gene into h ematopoietic stem cells is an attractive approach to the therapy of hu man beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required fo r a significant therapeutic effect. The discovery of the beta-locus co ntrol region (beta-LCR), organized in four major DNase I hypersensitiv e sites far upstream of the human beta-like globin gene cluster, provi ded a potential means to achieve a high level of expression of a linke d human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-t iter viruses with multiple rearrangements of the transmitted proviral; structures. We now describe how extensive mutagenesis of the transduc ed beta-globin gene, eliminating a 372 bp intronic segment and multipl e reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon inf ection of cell lines and bone marrow-repopulating cells. These optimiz ed vectors have enabled us to analyze the expression properties of var ious retrovirally transduced beta-LCR derivatives in dimethylsulfoxide -induced murine erythroleukemia cells and to achieve ratios of human b eta-globin/murine beta(maj)-globin mRNA, on a per gene basis, as high as 80%.